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VEGF modulates early heart valve formation.

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VEGF Modulates Early Heart
Valve Formation
Department of Molecular Biology, Hebrew University-Hadassah Medical School,
Jerusalem, Israel
Department of Pediatrics and Steele Memorial Children’s Research Center,
University of Arizona, Tucson, Arizona
Speciality Care Center, Veterans Administration Medical Center, Salt Lake City, Utah
Department of Pharmacology and Toxicology, College of Pharmacy,
University of Arizona, Tucson, Arizona
Although hypoxic and/or nutritional insults during gestation are believed to contribute to congenital heart defects, the mechanisms responsible
for these anomalies are not understood. Given the role vascular endothelial
growth factor (VEGF) plays in response to hypoxia, it is a likely candidate
for mediating deleterious effects of embryonic hypoxia. The ectopic or overproduction of endogenous factors such as VEGF may contribute to specific
heart defects. Here we compared hypoxia-induced precocious production of
VEGF during early heart valve development to normal VEGF production.
Mouse prevalvular cardiac endocardial cushions were explanted onto hydrated type I collagen gels under normoxic or hypoxic conditions. The extent
of transformation of cardiac endothelium into mesenchyme was inversely
correlated with the levels of VEGF during the various culture conditions. A
soluble VEGF antagonist confirmed that endogenous production of VEGF
was specific for blocking normal cushion mesenchyme formation. We further demonstrated that E10.5 endocardium retains the ability to transform
into cardiac mesenchyme in the absence of endogenous VEGF. Anat Rec
Part A 271A:202–208, 2003. © 2003 Wiley-Liss, Inc.
Key words: VEGF; hypoxia; development; heart; endocardial
The angiogenic response is essential for a variety of
physiological and pathological processes. The importance
of angiogenesis during heart development is demonstrated by a clinical association between congenital heart
defects and gestational hypoxia (DeSesso, 1987; Lueder et
al., 1995). VEGF is known to act in restoring hypoxic
mature tissue to normal oxygen homeostasis by inducing
the generation of new blood vessels (Dor and Keshet,
1997). Strict control of VEGF levels is required to control
the angiogenic response and prevent deleterious effects
(Lee et al., 2000; Isner, 2001). In embryonic mouse development, a two- to threefold increase in endogenous VEGF
production results in midgestation lethality (Miquerol et
al., 2000). These embryos exhibit cardiac defects, such as
overdeveloped trebeculae and coronary vessels, as well as
septation abnormalities. Conversely, the loss of one VEGF
allele results in early embryonic lethality due to cardiovascular defects (Carmeliet et al., 1996; Ferrara et al.,
1996). This haploinsufficient phenotype exhibits underdeveloped endocardial cushions and chamber malformations
in addition to impaired vascular development. In a controlled transgenic system, we demonstrated that produc-
Grant sponsor: NIH; Grant numbers: HLBI 63926; HL F32
10299; AHA 0230056; Grant sponsor: PANDA Foundation of Arizona.
*Correspondence to: Todd D. Camenisch, Department of Pharmacology and Toxicology, College of Pharmacy, University of
Arizona, 1703 E. Mabel St., Tucson, AZ 85721.
Received 15 May 2002; Accepted 10 October 2002
DOI 10.1002/ar.a.10026
Published online 28 January 2003 in Wiley InterScience
tion of VEGF a full day earlier (E9.5 vs. E10.5) results in
septal and valve defects arising from malformed endocardial cushion tissues (Dor et al., 2001). These findings,
which demonstrate a dependence on appropriate VEGF
production for normal valve formation and chamber septation, appear to correlate with septal defects observed
from hypoxia or exposure to air pollutants during early
gestation (Miao et al., 1988; Ritz et al., 2002). These results suggest that precocious increases in VEGF may prematurely terminate epithelial–mesenchymal transformation (EMT) in the endocardial cushions, and contribute to
chamber septation and valvular defects.
At present, it is not clear whether hypoxia induces the
production of physiologic levels of VEGF during cardiac
development, or excess VEGF production prior to endocardial cushion morphogenesis leads to septation defects. In
addition, it is unknown whether cessation of cardiac endothelial transformation is regulated exclusively by
VEGF, or there is a requirement for additional mediators.
At a specific time in development (E9.0 –E10.0), inductive
signals derived from the cardiac myocardium activate endothelial cells in these regions to undergo EMT. Cell
transformation within the endocardial cushions occurs as
a subset of endothelial cell hypertrophy, disengage cell–
cell adhesions, extend filopodia, and invade the extracellular matrix (Markwald et al., 1979; Eisenberg and Markwald, 1995). In the current study we used an in vitro
collagen gel invasion assay to reproduce the morphogenetic events of atrioventricular canal (AVC) morphogenesis (Runyan and Markwald, 1983; Camenisch et al., 2002).
In this context, endocardial cushion tissue from E9.5 and
E10.5 mouse embryos were explanted and examined in
normoxic and hypoxic conditions. Levels of VEGF were
correlated with mesenchymal formation and/or expansion
of the AVC endocardium. The results indicate that precocious production of physiologic levels of VEGF can be
induced by hypoxia during this critical stage of heart
development. These observations are the first to define the
normal events initiated by VEGF immediately following
EMT (⬃E10.5). VEGF acts to terminate the formation of
mesenchymal progenitors that are required for chamber
septation and cardiac valve formation.
AVC Explants
Mouse AVC endocardial cushion “explants” from wildtype “FVB” mouse embryos were isolated from timed fertilized female mice, and embryonic age was verified by
somite number. Experimental conditions were optimized
for mouse endocardial cushion cultures to obtain morphologic events similar to those in the established avian system (Camenisch et al., 2002). Briefly, M199 media was
used for gel casting in four-well microculture dishes
(Nalge Nunc, Naperville, IL). OPTI-MEM medium (Gibco
BRL, Rockville, MD) plus 0.01% ITS (Gibco-BRL) was
used to hydrate the polymerized type I collagen gels before
placement of the mouse AVC endocardial cushion tissue.
Mouse explants required 12 hr for attachment to the collagen gel surface at 37°C, 5% CO2 prior to the application
of 0.1 ml of M199 supplemented with 1% fetal calf serum
(Hyclone, Logan, UT), 50 U/ml penicillin, 50 ␮g/ml streptomycin, and 0.01% ITS (Gibco-BRL). Explants were cultured for 48 hr at 37°C, 5% CO2 under normoxic (21%),
hypoxic (10%), or anoxia conditions produced by a BBL
GasPak anaerobic system (Becton Dickinson, Sparks,
MD). For explants treated with sFlt (25 ng/ml), the recombinant chimeric receptor was immediately added at the
time the AVC was placed onto the gel surface. The extent
of endocardial migration or spread, and mesenchymal cell
invasion was assessed using differential interference contrast (DIC) optics, as previously described (Camenisch et
al., 2002; Dor et al., 2001). Briefly, the morphologic criterion used for endocardial cell classification was the appearance of rounded, polygonal cells on the collagen gel
surface with intact cell– cell junctions. The primary morphologic criterion for mesenchymal cell invasion was the
appearance of cells with characteristic stellate appearance
within the gel matrix (Runyan and Markwald, 1983). Images were captured using a Nikon microscope with
Spot image software (Diagnostic Instruments, Sterling
Heights, MI).
Analysis of VEGF
VEGF levels were determined from culture supernatants collected following 48 hr of incubation using a specific mouse VEGF assay (Oncogene Research Products
Inc., Boston, MA). Briefly, harvested supernatants from
three AVC explants per well were diluted 1:2 in buffer and
treated according to the manufacturer’s instructions for
the detection of VEGF, with levels calculated to pg/ml per
AVC explant. A minimum of six wells was used to calculate VEGF levels for each condition. A paired-sample ttest was used to compare VEGF concentrations between
experimental groups. A P-value ⬍ 0.05 between experimental groups and normoxic controls was considered significant.
Hypoxia and Post-transformation Isolated AVC
Explants Exhibit Reduced Mesenchymal
Cell Formation and Expansion of the
Cardiac Endothelium
Embryonic endocardial cushions isolated from the AVC
were cultured on type I collagen gels to recapitulate the
events of EMT during AVC morphogenesis (Runyan and
Markwald, 1983). Mouse AVC explants isolated prior to
cell transformation in vivo (E9.5) demonstrated EMT with
little to no endothelial cell expansion in the collagen gel
invasion assay (Fig. 1A) (Lakkis and Epstein, 1998; Camenisch et al., 2000). These mesenchymal cells extended
filopodia, and invaded the collagen matrix. In contrast,
AVC explants at the post-transformation stage (E10.5)
showed a substantial expansion of a cardiac endothelial
cell monolayer, and minimal transformation (Fig. 1B). The
cells typically maintained cell– cell borders and did not
invade the collagen gel. When E9.5 AVC explants were
exposed to hypoxic culture conditions, they exhibited a
phenotype similar to that of post-transformation E10.5
explants, with an expanded sheet of endothelial cells and
reduced mesenchymal cell formation (Fig. 1C). Anoxia
resulted in a similar post-EMT phenotype, but with increased cell toxicity (Semenza, 1999) and patches of endocardial cells (Fig. 1D) (Dor et al., 2001). Thus, hypoxia at
E9.5 appears to circumvent endocardial cushion EMT,
resulting in a “post-EMT” phenotype observed normally
with E10.5 AVC endocardial cushion tissue (Fig. 1C). This
response to hypoxia appears to override the normal transformation events required for endocardial cushion mesenchyme formation.
Fig. 1. AVC in vitro morphogenesis. AVC explants cultured for 48 hr
on hydrated type I collagen gels under the indicated conditions: (A)
normoxic E9.5 AVC, (B) normoxic E10.5 AVC, (C) E9.5 AVC under 10%
hypoxia, and (D) E9.5 AVC under anoxic conditions. There is a normal
EMT in part A, but EMT is drastically reduced or ablated in B–D. A
representative image is shown from a minimum of 10 explants for each
condition. M, myocardium. Scale bar in A ⫽ 100 ␮m, and scale bar in D
(for B–D) ⫽ 100 ␮m.
Post-EMT Phenotype Correlates With Increased
VEGF Production
VEGF, which is produced in response to hypoxia, antagonizes EMT. VEGF protein levels produced by E9.5 and
E10.5 AVC explants cultured under normoxic conditions
were compared to E9.5 AVC explants subjected to hypoxic
and anoxic environments. An elevation in VEGF concentration for endocardial cushions cultured under normoxic
conditions was observed between stages E9.5 and E10.5
(Fig. 2), supporting earlier in vivo findings of an increase
in VEGF mRNA during this period (Dor et al., 2001). Both
hypoxia and anoxia induced precocious VEGF production
in E9.5 AVC explants to levels significantly greater than
the endogenous levels normally produced at the E9.5
stage under normoxic conditions. The 10% O2 environment with E9.5 explants produced VEGF amounts comparable to naı̈ve E10.5 explants that were increased approximately 12-fold over control E9.5 AVC explants (43.12
and 45.1 vs. 3.35 pg/ml/AVC, respectively) (Fig. 2). These
elevated doses are within the in vivo range for VEGF
observed in embryonic hearts post-EMT (⬃E12.5)
(Miquerol et al., 2000). The elevated VEGF concentrations
detected from both E10.5 and hypoxic culture conditions
with E9.5 AVC explants correlates with the observed endothelial cell phenotype and decreased EMT (Fig. 1).
Fig. 2. VEGF production during in vitro AVC morphogenesis. VEGF
was detected in each experimental group, as described in the Methods
section. Reduced levels in anoxia and 5% O2 are speculated to reflect
reduced overall cellularity due to toxicity under these conditions (Semenza, 1999). Concentration of VEGF shown as pg/ml per AVC explant.
*P ⬍ 0.001 and **P ⬍ 0.01. Values were determined from independent
experiments as described in the Methods section.
Fig. 3. Neutralization of VEGF restores EMT in hypoxic and E10.5
AVC explants: (A) normoxic E10.5 AVC (control), (B) E10.5 with sFlt
inhibitor, (C) E9.5 AVC under 10% hypoxia, and (D) E9.5 AVC under 10%
hypoxia with sFlt inhibitor. Dashed line indicates footprint of removed
myocardium for ease of visualization. A representative image is shown
for each condition from observations of a minimum of eight AVC explants. Scale bar ⫽ 100 ␮m.
VEGF Is Sufficient for Inducing the Post-EMT
enchyme. Furthermore, our results indicate that VEGF is
a predominant mediator produced during embryonic hypoxia, and a key negative regulator of EMT within the AVC
endocardial cushions.
The remodeling events following EMT probably depend
on multiple factors, but VEGF may be a central switch
that demarcates this transition step away from EMT and
toward remodeling of the rudimentary valve tissue (Camenisch et al., 2002). In this regard, a soluble VEGF receptor 1 chimeric protein (sFlt), which functions as a soluble
antagonist of VEGF signaling (Gerber et al., 1999), was
used to determine whether the hypoxia-induced cellular
responses at E9.5 are specific to VEGF. When cultured in
the presence of the inhibitor sFlt, hypoxic E9.5 AVC cultures reverted to the normal E9.5 phenotype with mesenchymal cell formation and invasion (Fig. 3D vs. 3C), similarly to anoxic E9.5 cultures with sFlt (Dor et al., 2001).
We further examined the capacity of sFlt to attenuate the
post-EMT endocardial expansion observed in normal
E10.5 AVC explants (Fig. 3A). E10.5 AVC cultures treated
with sFlt demonstrated extensive mesenchymal cell formation and reduced endocardial outgrowth (Fig. 3B). This
phenotype is most similar to normoxic E9.5 AVC explants
(Fig. 1A), and shows that neutralizing VEGF promotes
EMT. These results define a critical temporal requirement
for the production of VEGF during early cardiac valve
formation. These observations also indicate that E10.5
AVC explants maintain the capacity to form cardiac mes-
VEGF is a critical factor during embryonic vasculogenesis and angiogenesis, much of which is thought to be
stimulated by temporal hypoxic environments. A recent
survey of embryonic tissues revealed that most organs,
including the heart, experience hypoxia during development (Lee et al., 2001). Cardiac chamber and vascular
endothelium malformations observed in HIF-1␣-deficient
mice also suggest that localized hypoxia most likely occurs
in embryos to induce vessel development related to organogenesis (Iyer et al., 1998; Ryan et al., 1998; Semenza et
al., 1999). However, no direct evidence has linked hypoxia
with induction of cardiac VEGF production during wildtype embryonic development. In this study, we defined the
developmentally programmed production of VEGF during
in vitro AVC endocardial cushion morphogenesis, as well
as the induction of VEGF by hypoxia. We detected a significant increase in the amount of VEGF protein produced
by the AVC between stages E9.5 and E10.5. During this
period, EMT produces mesenchymal cells that populate
the prevalvular endocardial cushions. Our current find-
Fig. 4. Schematic model comparing events during normal conditions
and hypoxic conditions for AVC endocardial cushion morphogenesis.
Proceeding from top to bottom, events are depicted chronologically
from E9.5 to E11.5. Precocious production of VEGF during hypoxia
decreases EMT, resulting in decreased remodeling and maintenance of
cushion volume. Text in bold during hypoxia conditions highlights dif-
ferences compared to the normoxia or normal events for EMT related to
endocardial cushion morphogenesis. M, myocardium; E, endocardium;
Ec, endocardial cells; ECM, extracellular matrix; Cm, cushion mesenchyme. Up arrows and down arrows denote increase and decrease,
respectively, and ⫹ indicates production or activity.
ings support a previous report (Dor et al., 2001) which
showed that VEGF mRNA is normally expressed in the
AVC following transformation events in vivo. Following
cardiac AVC mesenchyme formation, VEGF appears to
down-regulate EMT, and may stimulate the endocardium
of the AVC to proliferate. This capacity may serve to
maintain the integrity of endothelial cell junctions following EMT, and antagonize subsequent cell invasion into
the cardiac jelly (see Fig. 4).
The strict spatiotemporal production of VEGF during
heart morphogenesis emphasizes the necessity of regulating the concentrations of this growth factor during development. The loss of one VEGF allele results in early embryonic lethality due to cardiovascular defects (Carmeliet
et al., 1996). Likewise, a premature increase in endogenous VEGF production results in midgestation lethality
(Miquerol et al., 2000). These transgenic embryos exhibit
vasculature and cardiac abnormalities, such as septal defects, due to precocious induction of endocardial development. In this regard, we have demonstrated that a normal
increase in endogenous VEGF produced by myocardium of
the AVC in vitro terminates EMT. This level of production
is in accordance with a previous study by Miquerol et al.
(2000), who reported that a two- to threefold increase in
VEGF can elicit endothelial changes, and if mistimed can
have deleterious developmental impact. Our data show
that a premature elevation in VEGF, such as that induced
by hypoxia, results in decreased mesenchyme formation
along with an early angiogenic response by the endocardium of the developing AVC (Fig. 4). Furthermore, the
addition of exogenous VEGF165 (10 –100 ng/ml; Pepro
Tech, Rocky Hill, NJ) to normal E9.5 AVC explants reproduces the post-EMT phenotype (data not shown). Collectively, these findings indicate that strict spatiotemporal
regulation of VEGF production is required to establish
sufficient cardiac mesenchyme within the early valve tissue.
The current study emphasizes the importance of the
precise regulation of VEGF production during cardiovascular development, particularly in AVC morphogenesis.
VEGF is a well characterized mediator of responses to
hypoxia and other environmental insults (Carmeliet and
Jain, 2000). VEGF recruits new vessels to hypoxic tissues
to restore oxygen homeostasis in a variety of pathological
states and model systems (Dor and Keshet, 1997). In this
regard, congenital heart defects are more prevalent at
high altitudes (Miao et al., 1988), and increased cardiac
anomalies resulting from experimental hypoxia have been
shown in several animal models (Ingalls et al., 1952;
Clemmer and Telford, 1966; Jaffee, 1974). Although the
mechanisms mediating these effects have not been elucidated, congenital heart defects occurring from gestational
hypoxia likely depend on the onset and duration of the
hypoxic stress. Insufficient endocardial cushion mesenchyme formation resulting from premature VEGF activity
could contribute to structural heart anomalies, such as
atrial septal defects, which can arise from valvuloseptal
tissue deficiencies (Olson and Srivastava, 1996; Eisenberg
and Markwald, 1995).
Progress in understanding the molecular regulation of
endocardial cushion morphogenesis has been facilitated
by use of an in vitro collagen gel assay (Markwald et al.,
1981; Runyan and Markwald, 1983). In this regard, the
peptide growth factors TGF␤2, signaling through the type
III TGF␤ receptor, and TGF␤3, through the type II TGF␤
receptor, direct nonredundant signaling cascades to initiate EMT in the endocardial cushions of the AVC (Brown et
al., 1996, 1999; Boyer et al., 1999; Boyer and Runyan,
2001). TGF␤2 appears to mediate initial cell– cell separation of activated canal endocardium derived from chick
embryos, while TGF␤3 is essential for subsequent mesenchymal cell formation and invasion into the underlying
matrix. TGF␤3 is detected in mouse endocardial cushions
participating in coronary vasculogenesis and cardiac valve
remodeling after transformation events (⬃E11.5) (Baldwin, 1996; Camenisch et al., 2002). TGF␤3 increases
VEGF protein production in a dose-dependent manner
(Saadeh et al., 2000). In the current study, this timing of
TGF␤3 activity coincided with the production of VEGF,
which suggests that TGF␤3 may be one factor upstream of
AVC production of VEGF, or, conversely, VEGF may trigger TGF␤3 activity demarcating a shift in the morphogenetic events of EMT. The ability of E10.5 explants to
retain the capacity to undergo EMT emphasizes the induction power of VEGF to circumvent the signals that
promote mesenchyme formation. We have demonstrated
that VEGF mediates post-transformation responses by
endocardial cells within the embryonic AVC, and that
these events can be induced prematurely by exposure to
hypoxic conditions. Further investigations are warranted
to determine whether TGF␤3 and VEGF production is
functionally linked in the context of remodeling the cardiac cushions into heart valves.
We thank Dr. N. Ferrara (Genentech) for the generous
gift of the sFlt-Ig chimeric protein. We appreciate the
critical review of the manuscript by Dr. J. Schroeder, and
the assistance of Ms. Sharon Fleck with the manuscript
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